Previous studies suggest that enterovirus infections may initiate and accelerate beta-cell damage years before the clinical manifestation of type 1 diabetes. We have now analyzed the role of enterovirus infections in the initiation of autoimmunity in children who have tested positive for diabetes-associated autoantibodies in a prospective study starting at birth (the Finnish Diabetes Prediction and Prevention Study). The frequency of enterovirus infections was studied using both serology and testing for the presence of enterovirus RNA in the sera of 21 children who developed and retained autoantibodies and in 104 control subjects chosen from the same study cohort and matched for the time of birth, sex, and HLA alleles determining genetic diabetes susceptibility. Sample intervals were taken as basic units of follow-up, to which the observed number of infections was adjusted. Enterovirus infections were detected in 26% of sample intervals in the case subjects and in 18% of the sample intervals in the control children (P = 0.03). A temporal relationship between enterovirus infections and the induction of autoimmunity was found; enterovirus infections were detected in 57% of the case subjects during a 6-month follow-up period preceding the first appearance of autoantibodies compared with 31% of the matched control children in the same age-group (odds ratio 3.7, 95% CI 1.2-11.4). The frequency of adenovirus infections did not differ between the patient and control groups. Our data imply that enterovirus infections are associated with the development of beta-cell autoimmunity and provide evidence for the role of enteroviruses in the initiation of beta-cell destruction.